Investigation of TXNIP (thioredoxin-interacting protein) and TRX (thioredoxin) genes for growth-related traits in pigs

Investigation of TXNIP (thioredoxin-interacting protein) and TRX(thioredoxin) genes for growth-related traits in pigs

Mei Yu,1,2 Becky Geiger,3 Nader Deeb,3 Max F. Rothschild1

1Department of Animal Science and Center for Integrated Animal Genomics, Iowa State University, 2255 Kildee Hall, Ames Iowa, 50011,USA2Key Lab of Agricultural Animal Genetics, Breeding, and Reproduction of Ministry of Education, College of Animal Science andTechnology, Huazhong Agricultural University, Wuhan, 430070, People�s Republic of China3PIC/Genus plc, 100 Bluegrass Commons Blvd., Suite 2200, Hendersonville, Tennessee 37075, USA

Received: 20 November 2006 / Accepted: 22 January 2007

Abstract

It is well known that TRX and its endogenousinhibitor TXNIP help sustain the cellular reduction/oxidation balance in response to various stresses andboth play a crucial role in cell proliferation andgrowth. Five SNPs were found in TXNIP and theseallowed us to map it by linkage to SSC4. Three of theSNPs were used for association analyses in threecommercial pig populations (Duroc, Hampshire, andsynthetic line) with more than 1200 animals. Boththe single-marker and haplotype analyses revealedsignificant effects of TXNIP on hot carcass weight,test daily gain, and lifetime daily gain. TRX wasmapped on SSC1 but no significant associations withgrowth-related traits were found in the synthetic pigline in which the SNP was informative. Theexpression levels of TXNIP and TRX were then de-tected in two groups (fast growth and slow growth,respectively) with different genetic backgrounds forgrowth. Compared with the slow-growth group,TXNIP expression was significantly lower in thefast-growth group, whereas a marked increase inTRX expression was observed in fast-growth group.Our findings suggest that TXNIP has effects ongrowth-related traits in pigs and further investiga-tions will be necessary to elucidate the underlyingmechanisms involved.

Introduction

Thioredoxin-interacting protein (TXNIP) gene, alsotermed vitamin D3 up-regulated protein 1 (VDUP1)or thioredoxin-binding protein-2 (TBP-2) gene, wasoriginally found to be upregulated in HL-60 leuke-mia cells treated with 1,25-dihydroxyvitamin D3

(Chen and DeLuca 1994). Txnip has been mapped tomouse chromosome 3 band F2.2 (Ludwig et al.2001), while its human homolog is located onchromosome 1q21, a region that is frequently mu-tated or lost in cases of human cancers (Biecheet al. 1995; Medvedev et al. 1997; Keung et al.1998). TXNIP is located in the cytoplasm and is amultifunctional protein involved in suppression ofcell proliferation and growth and in apoptosis inresponse to oxidative stress. The role of TXNIP incell proliferation and growth was mainly evidentfrom the observations that TXNIP inhibits prolif-eration in a variety of cells and exhibits a tumor-suppressive effect in several types of cancer byarresting cells at the G0/G1 phase (Yang et al. 1998;Ikarashi et al. 2002; Schulze et al. 2002; Filby et al.2006). In alignment with these findings, theexpression of TXNIP has been found to be induceddramatically by various stresses or stimuli such asH2O2, glucose toxicity, anticancer and antiprolifer-ative agents, which in turn renders cells morevulnerable to oxidative stress and could lead toinduction of growth arrest and/or apoptosis (Junnet al. 2000; Takahashi et al. 2002; Wang et al. 2002;Minn et al. 2005).

To date, one of the currently known mecha-nisms underlying the effects of TXNIP is mediatedby inhibition of the reducing activity of thioredoxin(TRX) through direct interaction with the catalytic

Correspondence to: Max F. Rothschild; E-mail: [email protected]

DOI: 10.1007/s00335-007-9006-8 � Volume 18, 197�209 (2007) � � Springer Science+Business Media, LLC 2007 197

site of TRX (Nishiyama et al. 1999; Schulze et al.2002). TRX is the major component of the thiore-doxin system (thioredoxin, thioredoxin reductase,and NADPH), which plays a critical role in the reg-ulation of cellular reduction/oxidation (redox) bal-ance through the reversible oxidation of thioredoxinat two cysteine residues. The encoded gene of TRXhas been mapped on human chromosome 9 at band9q32 and on mouse chromosome 4 (Taketo et al.1994; Heppell-Parton et al. 1995). There is growingevidence suggesting that TRX has a role in protec-tion against cellular oxidative stress by maintainingseveral thioredoxin peroxidase enzymes in reduced,active forms and by inducing expression of otheroxidation defense enzymes (Watson et al. 2004;Yegorova et al. 2006). On the other hand, the effectsof TRX on cell proliferation promotion and apopto-sis inhibition have been extensively described.Although the mechanisms involved are multifac-eted, they could be explained, at least in part, bythe interaction of reduced TRX with those prolifer-ation- and apoptosis-associated proteins such asproliferation-associated gene (PAG) and apoptosissignal-regulating kinase 1 (ASK1) (Junn et al. 2000;Powis et al. 2001; Yoshioka et al. 2004). Most re-cently, several independent reports revealed thatTXNIP binds to reduced TRX to form a stabledisulfide-linked complex, which results in decreasedTRX-reducing activity and inhibited interaction ofTRX with other proteins, indicating that TXNIP actsas an endogenous inhibitor of TRX (Schulze et al.2002; Yoshioka et al. 2004; Patwari et al. 2006).Overexpression of TXNIP in various cells, includingvascular smooth muscle cells, cardiomyocytes, lungcells, and tumor cells, leads to reduced cellulargrowth accompanied by a decrease of TRX-reducingactivity and an increase of cellular oxidative stress(Butler et al. 2002; Schulze et al. 2002; Nishinakaet al. 2004; Yoshioka et al. 2004; Filby et al. 2006).Taken together, all these findings suggest a crucialrole for both TXNIP and TRX in redox-mediatedcontrol of cell proliferation and growth.

It has been widely accepted that the intracellularredox state is a critical regulator of various aspectsof cellular functions such as cellular prolifera-tion, activation or growth inhibition, and cell death(Yegorova et al. 2006). However, decades of stronggenetic selection for lean and fast growth in pigshave been linked to an increased prevalence ofmetabolic diseases and stress syndrome, which inturn may lead to oxidative stress (Brambilla et al.2002). It is of growing concern that increased oxida-tive stress could have substantial impact on ani-mal productivity and health (Brambilla et al. 2002;Sauerwein et al. 2005). Because thioredoxin and its

endogenous inhibitor TXNIP are well known to helpsustain the cellular reduction/oxidation balance inresponse to various stresses, we aimed to investigatethe possible involvement of the TXNIP and TRXgenes in growth-related traits in pigs. Related to thisobjective, we report (1) the isolation and character-ization of the porcine homolog for the human TXNIPgene; (2) the linkage mapping of both the TXNIP andthe TRX gene in a pig resource family; (3) theinvestigation of the effects of the two genes ongrowth-related traits examined in a pig resourcefamily and several commercial pig populations; andfinally (4) the examination of mRNA expressionlevels of the two genes in porcine skeletal musclefrom two groups of pigs with a different geneticbackground for growth.

Materials and methods

Animals and traits measured. This research wascarried out using a three-generation resource familyof a cross between the Berkshire and the Yorkshire(BY) pig breeds and three pig commercial lines. In theBY family, a total of 515 F2 animals were producedand were harvested in a commercial facility whenthey approached 115 kg. Traits measured in the BYresource family included four growth-related traitsrecorded on live animals, seven body compositiontraits, and a total of 28 meat quality traits. Detailsabout the family structure, management of pigs, andtrait measurements have been described by Maleket al. (2001). Three pig commercial lines (Duroc,Hampshire, and a synthetic line derived from severalbreeds including Pietrain, Duroc, Large White,and Landrace) were kindly provided by PIC USA(Hendersonville, TN) for use in this study. All ofthese lines have been maintained as closed popula-tions for at least 10 years (range = 11�29 years).Phenotypic data were collected at one commercialmeat packer from 2001 through 2004 according toNational Pork Producers Council guidelines. Traitspresented in this article were HCW, hot carcassweight (kg); LDG, lifetime daily gain (g/d); and TDG,test daily gain (g/d), which was used to measure thedaily body weight change of a pig from postnurseryto harvest.

Expression of both TXNIP and TRX genes wasdetected in skeletal muscle from animals in a com-mercial pig population, which was derived from thecross between a commercial line of Duroc sires anddams from a synthetic white line. The populationwas subdivided into two groups (EBVF and EBVS)according to the sires� estimated breeding value(EBV) for age at 125 kg (AGE125). The EBVFgroup included the most rapid growing pigs sired by

198 M. YU ET AL.: TXNIP AND TRX GENES IN PIGS

high-EBV-growth boars (n = 41 in this study) and theEBVS group consisted of the slowest-growing pigssired by low-EBV-growth boars (n = 40 in this study).The least-squares means of AGE125 of the offspringwere significantly different between the EBVF andthe EBVS groups (p < 0.0001).

PCR cloning of porcine genomic TXNIP geneand sequence analysis. We used the human TXNIPcDNA sequence (GenBank accession No.NM_006472) as a query for BLAST searching of theTIGR porcine expressed sequence tag (EST) database(http://www.tigr.org/tdb/tgi/ssgi) and retrievedtwo overlapping porcine ESTs (TIGR gene indexTC219061 and TC219069), which were able to beassembled into a contig sequence. Genomic DNAsequence of the porcine TXNIP gene was obtainedstarting with four sets of exonic primer, flankingexon�intron boundaries generated based on thecontig sequence assembled and the genomicsequence of human TXNIP (GenBank accession No.AB051901). The positions of the four fragmentsobtained (here named TXNIPe12, TXNIPe35, TXN-IPe57, and TXNIPe73) are shown in Fig. 1. Subse-quently, a new set of primers, located withinthe intron 1 and intron 3 regions for forward andreverse, respectively, were designed to bridge the gapbetween fragments TXNIPe12 and TXNIPe35.Therefore, the five overlapping genomic fragmentswere assembled into an incomplete genomicsequence of porcine TXNIP with the 5¢ upstreamregion from the exon 1 absent. While the presentwork was in progress, in May 2005, a 0.66X coveragepig genome survey was released to the GenBankdatabase by the Chinese�Danish pig sequencingconsortium (Wernersson et al. 2005). As a result ofthe approximately 3.84 million shotgun sequencesdeposited, we were able to carry out a PCR cloningstrategy to identify the 5¢ upstream region thatwe could not previously obtain by using primersdesigned based on the porcine EST sequences as wedescribed above. The human TXNIP sequence(GenBank accession No. AB051901) was used tosearch for homologous pig genomic sequences in thePig Genome Survey sequences database (http://www.animalgenome.org/blast/), and we found thatone of these homologs retrieved (gnl|ti|853095803)matched the 5¢ upstream region, including the pro-moter of the human TXNIP gene. Therefore, a pair offorward and reverse primers located in the piggenomic sequence (gnl|ti|853095803) and the previ-ously obtained exon 1 region were used to amplifythe 5¢ upstream region of porcine TXNIP ( includinga 713-bp region 5¢ to ATG translation start codon).The primer sequences are shown in Table 1.

To determine the transcription start site of theporcine TXNIP gene, the 5¢-RACE (rapid amplifica-tion of cDNA ends) technique was performedaccording to the manufacturer�s instructions (First-Choice� RLM-RACE kit, Ambion, Austin, TX). TheDNase I-treated total RNA (1 lg) used for 5¢-RACEwas isolated from porcine longissimus dorsi muscle(RNeasy� Midi kit, Qiagen, Valencia, CA). Afterfirst-strand cDNA synthesis, two reverse gene-spe-cific primers designed in the exon 1 region (5¢ g.s.outer: 5¢-AGGTAGTCCAAGGTCTGTTTGCAC-3¢and 5¢ g.s. inner: 5¢-GACTTCACACACCTCCAC-TATCAC-3¢) were used in association with the5¢-RACE outer and inner primers supplied with thekit, respectively (5¢-RACE outer: 5¢-GCTGATGGCGATGAATGAACACTG-3¢ and 5¢-RACE inner:5¢-CGCGGATCCGAACACTGCGTTTGCTGGCT-TTGATG-3¢) to amplify the porcine TXNIP 5¢ end.PCR products were subcloned into the pGEM-T easyvector (Promega, Madison, WI) and sequenced with a377 ABI automated DNA sequencer (Applied Bio-systems, Foster City, CA).

Alignment of those genomic fragments was usedto obtain a genomic sequence of the porcine TXNIPgene using the Sequencher software 3.0 (GeneCodes, Ann Arbor, MI). The two web softwareapplications, PROSCAN (promoter scan, http://www.thr.cit.nih.gov/molbio/proscan/) (Prestridge1995) and TESS (Transcription Element SearchSoftware, http://www.cbil.upenn.edu/tess/), wereused to search promoter elements and predict tran-scription factor binding sites.

Polymorphism screening and genotyping of theTXNIP and TRX genes. We screened for sequencevariants, the coding sequence, intron�exon bound-aries, and 5¢ upstream region plus part of the 3¢untranslated region (3¢ UTR) of TXNIP in differentpig lines or breeds. The entire region was covered bysix PCR fragments listed in Table 1 and shown inFig. 1. For TRX, four genomic fragments were iso-lated for polymorphism identification with theprimers listed in Table 1. Sequence variants wereidentified based on sequence comparisons and con-firmed by digestion of PCR products using restric-tion enzymes. Three of the five sequence variantsfound in TXNIP and one of the four sequence vari-ants found in TRX were considered for furtherassociation analyses because they were found to besegregating in the pig populations used in the studyand were able to be genotyped by the PCR restrictionfragment length polymorphism (RFLP) method.Information involved in the subsequent primers forapplying PCR-RFLP tests to genotype the polymor-phic sites, restriction enzymes used, and sizes of the

M. YU ET AL.: TXNIP AND TRX GENES IN PIGS 199

two different alleles for each locus are shown inTable 2. It should be noted that although the firstpolymorphic site of TXNIP listed in Table 2, T206C,can be recognized by restriction enzyme of CspC I,the forward primer was designed to induce anotherrestriction site using the enzyme of Apa I simply tocut the cost for further large-scale genotyping.

Linkage mapping of the TXNIP and TRXgenes. The previously described three-generationresource family generated by crossing Berkshire andYorkshire (BY) pig breeds was used for linkagemapping of the TXNIP and TRX genes (Malek et al.2001). Linkage analysis was performed by using two-point and multipoint analyses of CRIMAP version2.4 (Green et al. 1990) and the ‘‘BUILD’’ option wasused to obtain the sex-averaged linkage map.

Single-marker association analyses. Associa-tion analyses between single nucleotide polymor-phisms (SNPs) and growth-related traits wereperformed in the BY F2 population and commercialpig lines using the models:

(1) Associations in the BY F2 population:

Yijkl ¼ lþ SEXiþYEAR� SEASONjþGENEk

þ LITTERlþLIVE WEIGHTþ eijkl

(2) Single-marker analyses within the commerciallines (only females were used so sex was not in-cluded as an effect):

Yijk ¼ lþ SLAUGHTER DATEiþGENEjþ SIREk

þHOT CARCASS WEIGHTþ eijk

Table 1. PCR primers for obtaining the genomic DNA sequence of the porcine TXNIP gene

Fragmentname

Primer sequence (5¢-3¢)(forward/reverse) Primer locationa

Productsize (bp)

Primer indexsequence

TXNIP5e1 GGAATAAACGGTCGCCTCTA Putative promoterregion

928 gnl|ti|853095803

TAGCGCAGGTAGTCCAAGGT Exon 1 TXNIPe12TXNIPe12 GGTGATAGTGGAGGTGTGTGA Exon 1 743 TC219061

GAGGAAGCTCAAAGCCAAAC Exon 2 TC219061TXNIPe35 AGCCAGCCAACTCAAGAGAC Exon 3 757 TC219061

CAACTCGAAGGATGTTGCAG Exon 5 TC219061TXNIPin13 TCAACACTACAGGGCTTTGC Intron 1 693 TXNIPe12

CTGCTTTCCGTTCCTTTCC Intron 3 TXNIPe35TXNIPe57 GTCATCGGTCAGAGGCAAT Exon 5 644 TC219061

AAGTAGGTGGTGGCATGAAC Exon 7 TC219061TXNIPe73 TGACACAGATGGCTCTCAAG Exon 7 819 TC219069

TGGGAAGCTCACTCTAAACC 3¢ UTR TC219069TRXe1 CCGCTTCCATCTCTTTTACC 5¢ UTR 629 gnl|ti|842402996

TGCAGAACGCATCATCTC Intron 1TRXe2 GTTTGCCAGTTTAGATGTGG Intron 1 404 gnl|ti|784755869

ACAAGAGCAGAAGGTGGTT Intron 2TRXe4 GAGTGTGGAGCTGTCCCTTT Intron 3 510 gnl|ti|775672198

GTCGCTTCCGTTTCTTCC Intron 4TRXe5 TCACAGACACAGCTCACATCC Intron 4 662 gnl|ti|848873149

CCTGCTAGAACAAAGGCAAC 3¢ UTRaExonic and intronic regions were predicted according to the corresponding human TXNIP sequence.

Fig. 1. Schematic representation of theporcine TXNIP gene organization and theapproximate location of the fivepolymorphic sites found in this study. Sixoverlapping fragments used to generate thegenomic sequence of porcine TXNIP aslisted in Table 1 are also shown. TSS = thetranscription start site.


(3) Because no significant line-by-genotype interac-tions were found for growth and carcass traits,the across-line analysis was then performed toestimate the effects of TXNIP with model:

Yijkl ¼ lþ SLAUGHTER DATEiþGENEjþLINEk

þ SIRElþHOT CARCASS WEIGHTþ eijkl

where Y = growth-related traits, sex = fixed effect ofgender, year-season = fixed effect of season ofslaughter, slaughter date = fixed effect of day ofslaughter, gene = fixed effect of genotype, line =fixed effect of commercial line, litter = randomeffect of litter, sire = random effect of sire, liveweight = covariable of live weight, hot carcassweight = covariable of hot carcass weight. A value ofp < 0.05 was considered statistically significant.

Haplotype analyses. Haplotype analyses wereperformed within and across lines to investigate thecombined effects of the three substitutions (T206C,2173indelGT, and A3704G) found in the TXNIPgene. Haplotype construction and frequency esti-mation were performed by using an implementedexpectation-maximization algorithm (Excoffier andSlatkin 1995). The model used for estimation of thesubstitution effects of haplotypes is the same asdescribed in the previous paragraph. One variablewas included for each haplotype with a value )1, 0,or 1 corresponding to the pig having 0, 1, or 2 copiesof the haplotype in question. The haplotype substi-tution effects are presented as the deviations fromthe mean of all the haplotypes.

Quantitative real-time RT-PCR. The loin mus-cle tissue (longissimus dorsi) excised from eachanimal after slaughter was immediately treated withRNAlater (Ambion, Austin, TX) and stored at )80�Cfor RNA isolation. Total RNA was extracted usingthe RNeasy� Midi Kit according to the manufac-turer�s instructions (Qiagen, Valencia, CA). ThecDNA was synthesized from 1 lg of total RNA(DNase I-treated) with the SuperScript III First-Strand cDNA Synthesis SuperMix kit (Invitrogen,Valencia, CA). Quantitative real-time RT-PCR wasperformed using IQ� SYBR Green PCR Supermix(Bio-Rad Laboratories, Hercules, CA) in a MyiQTm

Single Color Real-Time PCR Detection System(Bio-Rad Laboratories). The primers for TXNIP weredesigned to flank intron 1 and to amplify a 230-bpcDNA. The expression levels of TXNIP were nor-malized to the endogenous RNA control RPL32(ribosomal protein L32) gene. The primer sequenceswere as follows: TXNIP sense: 5¢-CGACCCCGAAAT

ab

le2.

Poly

morp

his

ms

gen

oty

ped

inth

eporc

ine

TX

NIP

gen

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Poly

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ith

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tion

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Posi

tion

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seq

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ard

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Siz

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Rest

ricti

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en

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sc

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NIP

a

T206C

Pu

tati

ve

pro

mote

rre

gion

TT

TA

AT

AT

TT

CG

GG

AA

GC

CG

CC

CA

CG

GG

CC

130

Apa

I130

(T)

GA

CC

AA

TC

AG

CG

AA

GT

CG

CA

100

+30

(C)

2173in

del

GT

Intr

on

4G

GT

GT

CT

GT

CT

CT

GC

TC

GA

A240/2

38

Bsr

I240

(GT

(+))

AG

CT

TT

GG

GC

AC

CA

CA

AT

147

+91

(GT

()))

A3704G

3¢-U

TR

regi

on

AA

AT

GG

CC

TC

CT

GG

TG

TG

261

ApaL

I261

(A)

TG

GG

AA

GC

TC

AC

TC

TA

AA

CC

189

+72

(G)

TR

Xb

C1576T

Intr

on

3C

TG

AG

TT

GT

GG

GT

GC

CT

AA

C229

Tsp

509

I229

(C)

GT

CG

CT

TC

CG

TT

TC

TT

CC

170

+59

(T)

aP

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tion

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Gen

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991395.

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AGGTGTA-3¢ and antisense: 5¢-GAGGAAGCTCAAAGCCAAAC-3¢ (GenBank accession No.DQ991395); TRX sense: 5¢-GAGCAAGTACGCTTTTCAGG-3¢ and antisense: 5¢-ACCCACCTTCTGTCCCTTTT-3¢ (GenBank accession No.NM_214313); RPL32 sense: 5¢-TGGAAGAGACGTTGTGAGCAA-3¢ and antisense: 5¢-CGGAAGTTTCTGGTACACAATGTAA-3¢ (GenBank accessionNo. AY550039). All primers were optimized by meltcurve analyses to ensure that neither primer dimersnor other nonspecific products could be detected.The thermal profile consisted of 1 cycle at 95�C for10 min and then 35 cycles of 95�C for 30 sec, 59�Cfor 30 sec, and 72�C for 30 sec, followed by terminalelongation for 5 min at 75�C. All PCR reactions weredone in duplicate to achieve reproducibility. Thethreshold cycle (CT) value for each sample was cal-culated automatically by the Optical System version1.0 software. The duplicates for each sample wereaveraged. The DCT value was determined by sub-tracting the average RPL32 CT value from the aver-age of each gene CT value. Individual DCT values,which were used to evaluate the relative expressionlevels of TXNIP and TRX, respectively, were furthersubjected to statistical analyses by using SAS soft-ware (version 8; SAS, Inc., Cary, NC) with a model:

Yijkl¼ lþEBViþ SEXjþ SIREikþDAMiklþ eijkl

where Y = individual DCT values, EBV = fixed effectof EBV group, sex = fixed effect of gender, sire =random effect of sires nested within EBV group, anddam = random effect of dams nested within sire andEBV group. Genotype was considered in a pre-liminary analysis for TXNIP but was removed fromfinal analysis because the effect was found to be notsignificant on the relative expression level.

Results

PCR cloning and identification of the porcinegenomic TXNIP gene. Based on pig sequenceshomologous to the human TXNIP gene (two ESTsand a genomic fragment), we isolated a 3774-bp-longporcine genomic sequence. DNA sequencing andcomparative analysis based on the GenBank queryconfirmed it as the porcine TXNIP gene (Genbankaccession No. DQ991395). Analysis of this sequencerevealed a translation start codon (ATG) at nucleo-tide (nt) 714, and the nucleotide sequence around itmatched well the Kozak consensus sequence (A/GNNATGG), indicating that this is the proteintranslation start (Kozak 1987). A stop codon (TGA)was detected 2487 bp downstream from the ATG atnt 3203. By comparing the derived porcine sequence

with the human TXNIP gene, the exon/intronboundaries were determined. The porcine TXNIPgene is similar to the human gene in organizationand size and contains eight exons and seven introns.All exon/intron junctions are in agreement with theGT/AG consensus rule of the splice donor andacceptor site. To further define the 5¢ flanking region(714 bp upstream of the translation start codon ofATG), the web software PROSCAN, which canpredict promoter regions based on scoring homolo-gies with putative eukaryotic Pol II promotersequences (Prestridge 1995), was used to determinethe 5¢-UTR region and putative promoter elements.The analyses predicted an estimated transcriptionstart site 276 bp upstream of the ATG codon at nt438. To better characterize the 5¢ end for TXNIP, weperformed 5¢-RACE extension using total RNA iso-lated from muscle tissue. Sequence analysis of the5¢-RACE product allowed us to locate the putativetranscription start site to the position exactly mat-ched to that predicted by the software describedabove. In addition, a minimal promoter region of 251bp was recognized 13 bp upstream to the putativetranscription start site. The region includes theconsensus reverse and forward CCAAT boxes as wellas a TATA box, indicating that the porcine TXNIPgene has a consensus TATA-containing promoter.The positions of the putative CCAAT box and theTATA box are 99 bp and 23 bp upstream of theputative transcription start site, respectively, asshown in Fig. 2. Comparison of the porcine TXNIPpromoter sequence ()262 to +29 bp) obtained in thisstudy with those of human, chimpanzee, cow, andmouse revealed that the well-conserved CCAAT boxand TATA box and the transcription start site resideat approximately the same position among thesespecies studied .

Polymorphism screening. To find sequencevariants in the porcine TXNIP, we performed a sys-tematic search of the promoter and 5¢ UTR, thecoding sequence, intron/exon boundaries, and part ofthe 3¢ UTR in different pig lines or breeds. Fivesequence variants were found in this screening,although none of them is in the coding region. Allfive variants in relation to the gene structure ofporcine TXNIP are shown in the schematic overview(Fig. 1). The first variant, T173C, is located 2 bpaway from the putative minimal promoter region.The next two variants, T206C and G402C, weredetected within the putative minimal promoter. TheT206C variant is located 127 bp and 202 bp upstreamto the putative CCAAT box and the TATA box,respectively. The G402C variant was positioned be-tween the putative CCAAT and TATA boxes and


more closely to the TATA motif. The 2173indelGT,which is a 2-bp (GT) polymorphism, was found inintron 4, and the last variant detected in this study,A3704G, is within the 3¢-UTR region and located498 bp downstream to the TAG stop codon.

Of the five sequence variants discovered, theT173C substitution could not be recognized by acommercially available restriction enzyme sotherefore it was not considered further. Although theG402C substitution is within the putative minimalfunctional promoter, the SNP had to be excludedfrom further association analysis because of thefindings from the initial allele frequencies test thatthe substitution was less polymorphic in the BYresource family and in all the commercial linesstudied (data not shown). Therefore, the remainingthree sequence variants (T206C, 2173indelGT, andA3704G) were selected for further association anal-ysis in this study.

We sequenced the entire coding region of theTRX gene in the BY F3 individuals but no polymor-phic site was found (data not shown). Because of thepresence of two big introns (introns 1 and 3) within

TRX gene, only four separate fragments flankingexons 1, 2, 4, and 5, respectively, were isolated byPCR cloning (GenBank accession No. DQ991396).We sequenced the four exons, including their partialflanking intronic regions in the BY family founders.Four SNPs were discovered: two are in the 3¢ UTRand the other two are in introns 1 and 3. It is note-worthy that all four SNPs were found to be in com-plete linkage disequilibrium in the BY resourcefamily and the animals chosen from the Duroc,Hampshire, and synthetic lines (data not shown).The polymorphic site found in intron 3, which canbe recognized by the restriction enzyme Tsp509 I,was selected for further genotyping.

Linkage mapping. The polymorphic site ofT206C in TXNIP was found to be segregating in thefounders of the BY resource family. Therefore, usingthe T206C substitution, we mapped the TXNIP geneto the pig chromosome 4 (SSC4) linkage map by two-point linkage analysis. The multipoint analysis gen-erated a best sex-averaged map order of the genebetween linked markers (with distance between loci

Fig. 2. Sequence alignment of the minimal promoter region of the TXNIP gene: the pig (this study, GenBank accession No.DQ991395), the human (GenBank accession No. AB051901), the chimpanzee (GenBank accession No.AADA01261725),the cow (GenBank accession No. NW_235646), and the mouse (GenBank accession No. AF282825). The CAAT and TATAsignals are shadowed. A dash indicates a gap of sequence comparison. The putative transcription start site as detected by 5¢RACE in the pig TXNIP gene is marked with an inverted triangle and its location is designed as +1. Asterisks representconserved nucleotides among the five species. The inverted CCAAT box and the direct CACGAG repeat are boxed.


in Kosambi cM): SW45�21.0�SW512�1.7�TXNIP�15.1�SW2435. For TRX, the C1576T substitutionfound in intron 3, which affects the Tsp509 I restric-tion recognition site, was used to map the gene toSSC1 by two-point linkage analysis. The bestsex-averaged map order (with distance between lociin Kosambi cM) is S0331�3.3�MC4R�17.9�SW974�2.0�TRX�15.4�SW373.

Significant effects of TXNIP on growthtraits. We investigated the effects of the T206C and2173indelGT substitutions on growth and carcasstraits in the BY F2 population. The A3704G substi-tution was excluded from the analysis because of itsvery low level of polymorphism. Association analy-sis revealed that the T206C substitution has a sug-gestive effect on average daily gain on test, withallele 206C favorable for the trait (p = 0.06). How-ever, the 2173indelGT substitution did not showsignificant effects on any of the examined traits (datanot shown).

Individual substitution effects of the three poly-morphisms found in the TXNIP gene (T206C,2173indelGT, and A3704G) on growth-related traitswere analyzed within each individual commercialpig line and across the lines. Significant or sugges-tive trends toward differences were found among theT206C genotypes for HCW (p = 0.008), LDG (p = 0.1)in the synthetic line, and TDG in Hampshire(p = 0.006) and synthetic lines (p = 0.1). Except forthe effect found in the Hampshire line in which theheterozygous individuals had slightly higher TDGthan the two homozygous individuals, the effectswere in the same direction, with allele 206C thefavorable allele for all three traits (heavier HCW andhigher LDG and TDG) in the synthetic line. Theacross-line analysis revealed significant effects of theT206C substitution on HCW and TDG, and a sig-nificant additive effect on LDG was also detected(p = 0.036, Table 3). The substitution effects for allthree traits were in the same direction as those weobserved within the line analyses. For the 2173in-delGT, an effect close to significance was seen in thesynthetic line for HCW (p = 0.06) and a significanteffect for LDG (p = 0.03) was detected in theHampshire line pigs. The substitution effects werefound to be significant for LDG and TDG orapproached the significant level for HCW in theacross-line analysis. The allele GT(+) was found tobe consistently associated with the heavier HCWand higher LDG and TDG. The A3704G substitutionshowed the smallest substitution effect within thethree commercial pig lines investigated. Only sug-gestive associations were detected in the syntheticline for HCW (p = 0.09) and in Hampshire for TDGT

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(p = 0.06). However, the significant association withTDG was detected with allele A3704 being associ-ated with higher daily gain.

Haplotypes were constructed using the threepolymorphisms (T206C, 2173indelGT, and A3704G)in the TXNIP gene. Only four haplotypes were foundin the three commercial pig lines investigated in thisstudy (Table 4). In terms of haplotype frequency, theDuroc is the least polymorphic, with haplotype3 [206C � 2173GT(+)�3704A] the highest fre-quency (0.647) haplotype. Haplotype 1 [206T �2173GT(+)�3704G] has the highest frequency in theHampshire line, whereas in the synthetic line, hap-lotype 2 [206T � 2173GT())�3704G] was the mostfrequent. Haplotype 4 [206C � 2173GT(+)�3704G]was found to be the least frequent in all the threepopulations.

Figure 3 shows the estimates of the substitutioneffects of the TXNIP haplotypes for HCW, LDG, andTDG as the deviations from the average of the fourhaplotypes within and across lines. Within-lineanalysis revealed that haplotype 2 generally has thebiggest negative effects (lighter HCW and lower LDGand TDG) in most cases except for HCW and LDG inthe Duroc line. Across-line analysis results showedthat the effect of haplotype 2 on LDG was signifi-cantly different from the effects of haplotype 1(p = 0.047), haplotype 3 (p = 0.023), and haplotype 4(p = 0.008). In agreement with the effect of haplotype2 on LDG, with respect to TDG, haplotype 2 is sig-nificantly different than haplotype 3 (p = 0.001) andhaplotype 4 (p = 0.005). In addition, a suggestivedifference (p = 0.1) between haplotype 2 and haplo-type 1, with haplotype 2 consistently unfavorable forTDG, was observed in the across-line analysis. Sig-nificant substitution difference between haplotype 2and haplotype 4 with respect to HCW was alsorevealed (p = 0.001). Moreover, haplotype 4 wasfound to be associated with heavier HCW in theDuroc and synthetic populations and higher LDGand TDG in the Duroc population. When the data

from the three commercial lines were pooled, theanalysis revealed that haplotype 4 has significantlydifferent effects than haplotype 2, with haplotype 4associated with an increase in HCW, LDG, andTDG. Therefore, results from both the single-markerand haplotype analyses suggest the significant effectof TXNIP on growth-related traits in pigs.

Association analyses between TRX andgrowth-related traits. Association analysis in theBY F2 population revealed significant effects of theC1576T Tsp509 I polymorphism on growth andcarcass traits, including average backfat (p = 0.0006),

Table 4. Haplotype frequencies for the T206C, 2173indel-GT, A3704G in the TXNIP gene in the three commercialpig lines

Commercialpig lines No of animals

Haplotype frequencya

1 2 3 4

Duroc 438 0.015 0.234 0.647 0.104Hampshire 438 0.520 0.115 0.196 0.170Synthetic line 444 0.188 0.429 0.341 0.040All 1320 0.242 0.259 0.394 0.105aHaplotype 1: 206T � 2173GT(+)�3704G; Haplotype 2: 206T �2173GT())�3704G; Haplotype 3: 206C � 2173GT(+)�3704A;Haplotype 4: 206C � 2173GT(+)�3704G.

Fig. 3. Estimates of the substitution effects of the TXNIPhaplotypes for HCW, LDG, and TDG as deviations fromthe mean of the haplotypes within and across lines. (a, b)p < 0.05; (c, d) p < 0.01; (e, f) p < 0.005; (g, h) p < 0.001.


backfat at last rib (p = 0.0001), backfat at the lumbarregion (p = 0.0003), loin eye area (p = 0.0142), aver-age daily gain on test (p = 0.0427), and average dailygain to weaning (p = 0169). Allele C was associatedwith significant decreases in backfat thickness andincreases in loin muscle mass and average daily gain.We did not find the significant effects on thosegrowth and carcass traits in the synthetic populationinvestigated. However, it is noteworthy that the Callele frequency was 0.891 (n = 463) in the syntheticpopulation and 1.0 in both the Duroc (n = 24) andHampshire (n = 24) populations which had allundergone strong selection for growth.

Investigation of mRNA expression of the twogenes. Real-time RT-PCR was performed to assessthe expression levels of TXNIP and TRX in porcineskeletal muscle from the two groups (EBVF andEBVS) with different genetic background for growth.All three polymorphic sites found in TXNIP and theTRX C1576T variant were genotyped in the twogroups. No significant differences in the expressionof TXNIP were found among individuals bearingdifferent genotypes of any of the three polymorphicsites in TXNIP, and, in addition, the TRX C1576Tsubstitution did not segregate in the two groups. It isworth noting, however, that chi-squared analysisdetected significant differences in the allele fre-quencies of TXNIP T206C between the EBVF andEBVS groups (p = 0.007), with allele C the most fre-quent in the EBVF group. In addition, TXNIPexpression was significantly 1.5-fold lower in theEBVF group which consisted of pigs sired by thehigh-EBV-growth boars compared with the EBVSgroup which comprised pigs sired by the low-EBV-growth boars (p = 0.023) (Fig. 4A). On the con-trary, we found a marked 1.6-fold increase in TRXexpression in the EBVF group compared with theEBVS group (p = 0.0002) (Fig. 4B).

Discussion

In this study, in order to provide relationships ofsequence variants in TXNIP and TRX genes with

their functional changes, we obtained a 3774-bp-longporcine genomic sequence from a combination ofPCR cloning and 5¢ RACE and which was nearly theentire TXNIP genomic sequence with only the par-tial 3¢ UTR being isolated. The current available piggenomic sequence allowed us to isolate the5¢-flanking region of the TXNIP gene, in which theputative promoter region was deduced. Promoteranalysis revealed a number of putative binding sitesfor transcription factors, including a TATA element,two CCAAT boxes (including an inverted onelocated 411 bp upstream of the ATG codon,DQ991395), and several Sp1 factors. The invertedCCAAT box has been determined to be the tran-script factor NF-Y binding site and critical for thedirect induction of the human TXNIP gene promoterin several transformed cell lines by SAHA (sube-roylanilide hydroxamic acid) and which is known toinduce growth arrest and/or apoptosis of manytumor types in vitro and in vivo (Butler et al. 2002).Aside from the inverted CCAAT box, a perfect directrepeat—CACGAG spaced by 5 bp—was foundbetween the TATA and CCAAT boxes (starts 363 bpupstream of the coding region, DQ991395) (Fig. 2).TESS predicted that it is a putative binding site forthe upstream stimulation factor (USF). Ludwig et al.(2001) has described the direct repeat of CACGAG asa USF/MLTF repeat (major late transcription factorrepeat) in the mouse txnip gene. Furthermore, Minnet al. (2005) provided biological evidence that it is anE-box repeat carbohydrate response element (ChoRE)in the human TXNIP gene promoter conferring theglucose responsiveness observed in b-cells. Giventhat those two elements in the TXNIP gene pro-moter are well conserved in pig, human, bovine,chimpanzee, and mouse by comparative sequenceanalyses (Fig. 2), it is possible, therefore, that the tworegions may have common regulatory mechanismsin pigs and other species investigated. Molecularanalysis will be necessary to determine if theyindeed play a role in regulation of TXNIP geneexpression in pigs.

Association analyses of the three individualpolymorphisms of the TXNIP gene in the commer-

Fig. 4. Expression levels of (A) TXNIP and (B)TRX in skeletal muscle from the fastest-growing pigs sired by high-EBV-growth boars(EBVF) compared with the slowest-growing pigssired by low-EBV-growth boars (EBVS)measured by real-time RT-PCR.


cial pig populations revealed that all of them weresignificantly associated with at least one of thegrowth-related traits investigated in this study. TheT206C substitution, which is located in the putativepromoter region, was found to have significantadditive effects on all three traits, and a similar effecton average daily gain on test was also detected in theBY F2 population with allele 206C always thefavorable allele. The 2173indelGT polymorphismwas also found to have significant additive effects onall three traits in the commercial pig populations,whereas the same effects were not observed in theBY F2 population. However, the A3704G substitu-tion showed a small effect on TDG.

The haplotype analysis revealed important dif-ferences between the effects of haplotypes 2 and 4 ofthe TXNIP gene on HCW, LDG, and TDG in theacross-line analyses. For all traits, haplotype 2 wasthe unfavorable haplotype and it was associated withlighter hot carcass weight, lower lifetime daily gainand lower test daily gain. Haplotype 2 differs fromhaplotype 4 at the first two positions (T206C and2173indelGT) and is the only haplotype that con-tains 2173GT()). However, no significant differencesbetween the effects of haplotypes 2 [206T �2173GT())�3704G] and 1 [206T � 2173GT(+)�3704G] were found for any of the growth traitsinvestigated in the across-line analyses. Thesehaplotypes differ only at the position of the 2173in-delGT variant and it is unlikely that the 2173indel-GT variant makes an important contribution to thehaplotype substitution effects. In addition, for TDG,a highly significant difference (p = 0.001) wasobserved between haplotypes 2 and 3, which differ atall three variants, implying that A3704G also makesa small contribution to the haplotype substitutioneffects. Haplotype 1 [206T � 2173GT(+)�3704G] andhaplotype 4 [206C � 2173GT(+)�3704G] did notdemonstrate a significant difference compared withhaplotype 3 [206C � 2173GT(+)�3704A], whereas asignificant difference between the effects of haplo-type 1 and haplotype 4 (differs only at the T206Cvariant) on HCW was revealed (Fig. 3). Thus, theassociation analyses indicated the presence of thethree variants in the TXNIP gene with different sizesof effects on growth traits in pigs. Although wecannot attribute all of the differences to the T206Cvariant, results from both the single-substitutionand haplotype association analyses in a large sampleof the pigs provided evidence that, of the threevariants, the T206C substitution in the TXNIP genewas associated with the most significant and largestdifferences in growth traits.

We mapped the TXNIP gene to SSC4 in the QTLregions for average daily gain on test and loin eye

area detected in the BY family (Malek et al. 2001).This could explain in part the suggestive effect of theTXNIP gene on average daily gain on test observed inthe BY F2 population. A search of the pig QTLdatabase (PigQTLdb, http://www.animalge-nome.org/QTLdb) revealed the presence in otherpopulations of QTL for growth and carcass traits,including body weight at slaughter, cold carcassweight, average daily gain, backfat thickness, andlean mass, in the region where the TXNIP gene wasmapped (Knott et al. 1998; Perez-Enciso et al. 2000;Bidanel et al. 2001; Cepica et al. 2003; Geldermannet al. 2003). However, given that we observed theinconsistent effects of the three substitutions amongthe three lines in both the single-marker and hap-lotype analyses, the three substitutions investigatedare likely not causal but may be in linkage disequi-librium with causative genetic variation withinparticular populations.

More recently, increasing attention has beenpaid toward the central role of the intracellular re-dox state in cell signaling, gene expression changes,and proliferation. Therefore, it is critical to main-tain the intracellular redox balance which dependson, to a large extent, the levels of reactive oxygenspecies (ROS) produced during metabolism and theactivity of the antioxidant system that scavengesthe ROS (Dietz et al. 2004; Jackson et al. 2005;Menon et al. 2006). Accumulating evidence hasdocumented the dual functions of one of the redox-active components, thioredoxin, and its endogenousinhibitor, TXNIP, in regulating cellular redox bal-ance and in controlling cell proliferation andgrowth (Yoshioka et al. 2004; Yegorova et al. 2006).In this study we found that both genes TXNIP andTRX are differentially expressed between the twopig groups with different genetic backgrounds forgrowth. On the other hand, we also observed theinverse relationship between the expression levelsof TXNIP and TRX in the two pig groups. There areseveral possible explanations for our findings. First,one of the well-characterized functions of thiore-doxin is the thiol-reducing activity required forprotecting the cell against cytotoxic oxidative stress(Powis et al. 2001; Butler et al. 2002). A decrease inthe level of TXNIP expression accompanied by aconcomitant increase in the level of TRX expres-sion may result in decreasing cellular oxidativestress, while an increase in expression of TXNIPfollowed by decreased expression levels of TRX maybe responsible for the enhanced oxidative damage.Second, thioredoxin is believed to be involved inthe redox regulation of a number of apoptosis-re-lated transcription factors, including PAG andASK1 (Junn et al. 2000; Powis et al. 2001; Yoshioka


et al. 2004; Yamawaki et al. 2005). Under condi-tions of oxidative stress, the thioredoxin confor-mation is altered and thereby leads to thedissociation of ASK1 from thioredoxin inhibition.On the other hand, TXNIP has been reported toinhibit cell growth by competing with PAG andASK1 for binding to TRX, which subsequently re-sults in promoting apoptosis. Third, growing evi-dence has been acquired that has shown TXNIPpossesses tumor-suppressive activity in severaltypes of cancer, such as breast, gastrointestinal (GI),and lung cancers, and ectopic expression of TXNIPresults in suppression of cell proliferation alongwith cell cycle arrest at the G1 phase (Yang et al.1998; Ikarashi et al. 2002; Filby et al. 2006). Fur-thermore, Matsushima et al.(2006) reported that theincreased expression of TXNIP and decreasedexpression of TRX might lead to disuse of skeletalmuscle and atrophy in the rat. We failed to observethe significant difference of the TXNIP expressionlevels between individuals bearing different geno-types in all three polymorphic sites. However, itremains possible that other sequence variants maystill exist within the regulatory regions that havealready been identified in other species (Ludwiget al. 2001; Butler et al. 2002; Minn et al. 2005).

In addition, we did not observe the same effectsof TRX on growth and carcass traits in the com-mercial pig lines as the results from the BY popula-tion. The most extensively investigated gene onSSC1 is the melanocortin 4 receptor (MC4R), whichhas been documented to be associated with growthand feed intake and recently was suggested to be apositional candidate gene for a fat/meat QTL (Kimet al. 2000; Bruun et al. 2006). Although the TRXgene was mapped 19.9 cM away from MC4R, bothgenes are located in the QTL interval that affectsfatness detected in the BY population (data notshown). The observed effects are possibly due to thelinkage disequilibrium that exists in the F2 popula-tion. However, given the finding that the frequencyof allele C, which is favorable for carcass traits in theBY population, is almost fixed in the three com-mercial populations investigated, we cannot fullyexclude the TRX effects on growth.

Our study is the first to provide an initial esti-mate of the association of the TXNIP gene withgrowth-related traits in a large sample of pigs. Inaddition, we found that TXNIP and TRX genes aredifferentially expressed between the two pig groupswith different genetic backgrounds for growth.Taken together, our results suggest a possible asso-ciation of the TXNIP and TRX genes with growth inpigs. Further research is needed, however, to eluci-date the nature of the association.

Acknowledgments

This work was funded in part by Genus, PIC USA,the Iowa Agriculture and Home Economics Experi-ment Station, and State of Iowa and Hatch funds.Technical assistance from Ms. Kimberly Glenn andDrs. S. Lonergan and C. Stahl is appreciated.

References

1. Bidanel JP, Milan D, Iannuccelli N, Amigues Y,Boscher MY, et al. (2001) Detection of quantitativetrait loci for growth and fatness in pigs. Genet Sel Evol33, 289�309

2. Bieche I, Champeme MH, Lidereau R (1995) Loss andgain of distinct regions of chromosome 1q in primarybreast cancer. Clin Cancer Res 1, 123�127

3. Brambilla G, Civitareale C, Ballerini A, Fiori M,Amadori M, et al. (2002) Response to oxidative stressas a welfare parameter in swine. Redox Rep 7, 159�163

4. Bruun CS, Jørgensen CB, Nielsen VH, Andersson L,Fredholm M (2006) Evaluation of the porcine melano-cortin 4 receptor (MC4R) gene as a positional candidatefor a fatness QTL in a cross between Landrace andHampshire. Anim Genet 37, 359�362

5. Butler LM, Zhou X, Xu WS, Scher HI, Rifkind RA,et al. (2002) The histone deacetylase inhibitor SAHAarrests cancer cell growth, up-regulates thioredoxin-binding protein-2, and down-regulates thioredoxin.Proc Natl Acad Sci USA 99, 11700�11705

6. Cepica S, Stratil A, Kopecny M, Blazkova P, Schroffel JJr, et al. (2003) Linkage and QTL mapping for Susscrofa chromosome 4. J Anim Breed Genet 120, 28�37

7. Chen KS, DeLuca HF (1994) Isolation and character-ization of a novel cDNA from HL-60 cells treated with1,25-dihydroxyvitamin D-3. Biochim Biophys Acta1219, 26�32

8. Dietz KJ, Scheibe R (2004) Redox regulation: anintroduction. Physiol Plant 120, 1�3

9. Excoffier L, Slatkin M (1995) Maximum-likelihoodestimation of molecular haplotype frequencies in adiploid population. Mol Biol Evol 12, 921�927

10. Filby CE, Hooper SB, Sozo F, Zahra VA, Flecknoe SJ,et al. (2006) VDUP1: a potential mediator of expan-sion-induced lung growth and epithelial cell differen-tiation in the ovine fetus. Am J Physiol Lung Cell MolPhysiol 290, 250�258

11. Geldermann HE, Muller G, Moser G, Reiner H, Bart-enschlager S, et al. (2003) Genome-wide linkage andQTL mapping in porcine F2 families,. J Anim BreedGenet 120, 363�393

12. Green P, Falls K, Crooks S (1990) Documentation forCRIMAP, version 2.4. St Louis, MO, WashingtonUniversity School of Medicine

13. Heppell-Parton A, Cahn A, Bench A, Lowe N, LehrachH, et al. (1995) Thioredoxin, a mediator of growthinhibition, maps to 9q31. Genomics 26, 379�381

14. Ikarashi M, Takahashi Y, Nagata T, Ishii Y, Ishikawa K,et al. (2002) Vitamin D3 up-regulated protein 1 (VDUP1)


expression in gastrointestinal cancer and its relation tostage of disease. Anticancer Res 22, 4045�4048

15. Jackson MJ (2005) Reactive oxygen species and redox-regulation of skeletal muscle adaptations to exercise.Philos Trans R Soc Lond B Biol Sci 360, 2285�2291

16. Junn E, Han SH, Im JY, Yang Y, Cho EW, et al. (2000)Vitamin D3 up-regulated protein 1 mediates oxidativestress via suppressing the thioredoxin function. JImmunol 164, 6287�6295

17. Keung YK, Yung C, Wong JW, Shah F, Cobos E, et al.(1998) Unusual presentation of multiple myelomawith ‘‘jumping translocation’’ involving 1q21. A casereport and review of the literature. Cancer Genet Cy-togenet 106, 135�139

18. Kim KS, Larsen N, Short T, Plastow G, Rothschild MF(2000) A missense variant of the porcine melanocortin4 receptor (MC4R) gene is associated with fatness,growth, and feed intake traits. Mamm Genome 11,131�135

19. Knott SA, Marklund L, Haley CS, Andersson K, DaviesW, et al. (1998) Multiple marker mapping of quanti-tative trait loci in a cross between outbred wild boarand large white pigs. Genetics 149, 1069�1080

20. Kozak M (1987) An analysis of 5V-noncoding se-quences from 699 vertebrate messenger RNAs. Nu-cleic Acids Res 15, 8125�8148

21. Ludwig DL, Kotanides H, Le T, Chavkin D, Bohlen P,et al. (2001) Cloning, genetic characterization, andchromosomal mapping of the mouse VDUP1 gene.Gene 269, 103�112

22. Malek M, Dekkers JCM, Hakkyo KL, Baas TJ, Roths-child MF (2001) A molecular genome scan analysis toidentify chromosomal regions influencing economictraits in the pig: I. Growth and body composition.Mamm Genome 12, 630�636

23. Matsushima Y, Nanri H, Nara S, Okufuji T, Ohta M,et al. (2006) Hindlimb unloading decreases thioredox-in-related antioxidant proteins and increases thiore-doxin-binding protein-2 in rat skeletal muscle. FreeRadic Res 40, 707�714

24. Medvedev A, Chistokhina A, Hirose T, Jetten AM(1997) Genomic structure and chromosomal mappingof the nuclear orphan receptor ROR gamma (RORC)gene. Genomics 46, 93�102

25. Menon SG, Goswami PC (2006) A redox cycle withinthe cell cycle: ring in the old with the new. Oncogene26, 1101�1109

26. Minn AH, Hafele C, Shalev A (2005) Thioredoxin-interacting protein is stimulated by glucose through acarbohydrate response element and induces beta-cellapoptosis. Endocrinology 146, 2397�2405

27. Nishinaka Y, Nishiyama A, Masutani H, Oka S,Ahsan KM, et al. (2004) Loss of thioredoxin-bindingprotein-2/vitamin D3 up-regulated protein 1 in humanT-cell leukemia virus type I-dependent T-cell trans-formation: implications for adult T-cell leukemialeukemogenesis. Cancer Res 64, 1287�1292

28. Nishiyama A, Matsui M, Iwata S, Hirota K, MasutaniH, et al. (1999) Identification of thioredoxin-bindingprotein-2/vitamin D(3) up-regulated protein 1 as a

negative regulator of thioredoxin function andexpression. J Biol Chem 274, 21645�21650

29. Patwari P, Higgins LJ, Chutkow WA, Yoshioka J, LeeRT (2006) The interaction of thioredoxin with txnip:evidence for formation of a mixed disulfide by disul-fide exchange. J Biol Chem 281, 21884�21891

30. Perez-Enciso M, Clop A, Noguera JL, Ovilo C, Coll A,et al. (2000) A QTL on pig chromosome 4 affects fattyacid metabolism: evidence from anIberian by Landraceintercross. J Anim Sci 78, 2525�2531

31. Powis G, Montfort WR (2001) Properties and biologicalactivities of thioredoxins. Annu Rev Pharmacol Toxi-col 41, 261�295

32. Prestridge DS (1995) Predicting Pol II promotersequences using transcription factor binding sites. JMol Biol 249, 923�932

33. Sauerwein H, Schmitz S, Hiss S (2005) The acute phaseprotein haptoglobin and its relation to oxidative statusin piglets undergoing weaning-induced stress. RedoxRep 10, 295�302

34. Schulze PC, De Keulenaer GW, Yoshioka J, Kassik KA,Lee RT (2002) Vitamin D3-upregulated protein-1(VDUP-1) regulates redox-dependent vascular smoothmuscle cell proliferation through interaction withthioredoxin. Circ Res 91, 689�695

35. Takahashi Y, Nagata T, Ishii Y, Ikarashi M, IshikawaK, et al. (2002) Up-regulation of vitamin D3 up-regu-lated protein 1 gene in response to 5-fluorouracil incolon carcinoma SW620. Oncol Rep 9, 75�79

36. Taketo M, Matsui M, Rochelle JM, Yodoi J, Seldin MF(1994) Mouse thioredoxin gene maps on chromosome4, whereas its pseudogene maps on chromosome 1.Genomics 21, 251�253

37. Wang Y, De Keulenaer GW, Lee RT (2002) Vita-min D(3)-up-regulated protein-1 is a stress-responsivegene that regulates cardiomyocyte viability throughinteraction with thioredoxin. J Biol Chem 277,26496�26500

38. Watson WH, Yang X, Choi YE, Jones DP, Kehrer JP(2004) Thioredoxin and its role in toxicology. ToxicolSci 78, 3�14

39. Wernersson R, Schierup MH, Jørgensen FG, GorodkinJ, Panitz F, et al. (2005) Pigs in sequence space: A 0.66Xcoverage pig genome survey based on shotgunsequencing. BMC Genomics 6, 70

40. Yamawaki H, Berk BC (2005) Thioredoxin: a multi-functional antioxidant enzyme in kidney, heart andvessels. Curr Opin Nephrol Hypertens 14, 149�153

41. Yang X, Young LH, Voigt JM (1998) Expression of avitamin D-regulated gene (VDUP-1) in untreated andMNU-treated rat mammary tissue. Breast Cancer ResTreat 48, 33�44

42. Yegorova S, Yegorov O, Lou MF (2006) Thioredoxininduced antioxidant gene expressions in human lensepithelial cells. Exp Eye Res 83, 783�792

43. Yoshioka J, Schulze PC, Cupesi M, Sylvan JD,MacGillivray C, et al. (2004) Thioredoxin-interact-ing protein controls cardiac hypertrophy throughregulation of thioredoxin activity. Circulation 109,2581�2586


Documents

Investigation of TXNIP (thioredoxin-interacting protein) and TRX (thioredoxin) genes for growth-related traits in pigs